短小芽孢杆菌脂肪酶基因的克隆、表达及酶学性质研究

生物技术通报 ›› 2014, Vol. 0 ›› Issue (4): 132-138.

短小芽孢杆菌脂肪酶基因的克隆、表达及酶学性质研究

苏二正¹, 吴向萍², 高蓓², 魏东芝²

(1. 南京林业大学轻工科学与工程学院酶与发酵工程实验室，南京 210037；
2. 华东理工大学生物反应器工程国家重点实验室鲁华生物技术研究所，上海 200237)

收稿日期:2013-12-23 出版日期:2014-04-29 发布日期:2014-04-29
作者简介:苏二正，男，博士，副教授，研究方向：分子酶学与生物催化；E-mail：ezhsu@njfu.edu.cn
基金资助:
国家高技术研究发展计划(“863”计划)(2012AA020403)，国家自然科学基金项目(31201296/C200101)，生物反应器工程国家重点实验室开放课题，南京林业大学引进高层次人才科研基金项目，江苏高校优势学科建设工程资助项目

Gene Cloning. Expression and Characterization of the Lipase from Bacillus pumilus S6

Su Erzheng¹, Wu Xiangping², Gao Bei², Wei Dongzhi²,

(1. Enzyme & Fermentation Technology Laboratory，College of Light Industry Science and Engineering，Nanjing Forestry University，Nanjing 210037；
2. State Key Laboratory of Bioreactor Engineering，New World Institute of Biotechnology，East China University of Science and Technology，Shanghai 200237)

Received:2013-12-23 Published:2014-04-29 Online:2014-04-29

摘要/Abstract

摘要： 从土壤样品中通过筛菌过程获得一株产脂肪酶的菌株，经16S rRNA 鉴定属于短小芽孢杆菌，根据已报道的来源于芽孢杆菌的脂肪酶基因设计引物，克隆获得其全长基因，命名为lipS6，大小为648 bp，编码215个氨基酸，经比对其与已报道的脂肪酶基因B26 有96%的同源性。构建重组表达质粒pET32a-lipS6，在大肠杆菌中实现了可溶表达，酶活达到2 000 U/mL。重组脂肪酶LipS6的最适温度为30℃，最适pH为9，低浓度的Ca²⁺与Mn²⁺对其有很明显的激活作用，疏水性有机溶剂对其毒害作用小，在正己烷体系中可以催化酯化反应，最适酯化底物酸为十四酸，底物醇为正丁醇。

关键词: 脂肪酶, 短小芽孢杆菌, 基因克隆, 重组表达, 性质

Abstract: A lipase-producing strain was screened from the soil samples. It was identified as Bacillus pumilus by the 16S rRNA method. According to the reported lipase genes from Bacillus, the primers were derived and the full-length gene was cloned, named “lipS6”, which had the size of 648 bp, encoding 215 amino acids. “lipS6” held 96% homology with the reported lipase gene B26. Recombinant plasmid pET32a-lipS6 was construced and expressed in E. coli. “lipS6”could be expressed in soluble form, and the lipase activity reached 2 000 U/L. The optimum temperature of recombinant lipase LipS6 was 30℃, and the optimum pH was 9. Low concentrations of Ca²⁺ and Mn²⁺ could activate the activity of lipase LipS6. The harmful effect of hydrophobic organic solvent on the lipase LipS6 was little. LipS6 could catalyze the esterification reaction in the n-hexane system, with the myristic acid and n-butanol as the optimum substrates. These properties show that LipS6 can be used in the fields of pulp and paper, leather, textiles and bioenergy.

Key words: Lipase, Bacillus pumilus, Gene cloning, Recombinant expression, Characterization

苏二正, 吴向萍, 高蓓, 魏东芝. 短小芽孢杆菌脂肪酶基因的克隆、表达及酶学性质研究[J]. 生物技术通报, 2014, 0(4): 132-138.

Su Erzheng, Wu Xiangping, Gao Bei, Wei Dongzhi,. Gene Cloning. Expression and Characterization of the Lipase from Bacillus pumilus S6[J]. Biotechnology Bulletin, 2014, 0(4): 132-138.

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